The longterm goal of the proposed research is to determine how genetic regulatory systems are determined by the joint contribution of their developmental and evolutionary histories. The Drosophila glucose dehydrogenase gene (Gld) will be used as a model system to study these determinants of gene regulation because Gld's tissue-specific regulation in the adult reproductive tract is highly variable among a variety of species in the genus Drosophila. The research strategy will be to use a combination of in vitro mutagenesis and gene transformation to critically test hypotheses regarding the role of cis-elements and trans-acting factors responsible for species' differences in developmental expression of GLD. The specific aims are largely focused on elucidating the regulatory factors underlying GLD expression in the reproductive organs of both males and females, and, in parallel, identifying regulatory differences between species. Since the sex- determination genes tra and tra-2 are required for the unique tissue- specific pattern of GLD expression in D. melanogaster, the Gld regulatory elements that respond to them will be mapped. Misregulation of tra and tra-2 in D. melanogaster leads to ectopic patterns of GLD expression in the reproductive tract that mimic normal patterns seen other Drosophila species. A hypothesis that other species' Gld genes differ in their response to the sex-determination genes will be tested. A newly devised genetic method for identifying genes that encode tissue specific transcription factors will be used to isolate such genes that regulate Gld. Knowledge of gene function alone provides insufficient and sometimes misleading information for understanding why genes are expressed in certain tissues. The proposed research should provide a deeper understanding of how evolution and development affect gene regulation thereby increasing our understanding of how perturbations in genetic regulatory systems can result in human genetic diseases.